Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Primers amplify adults but not immatures?!

Primers amplify adults but not immatures?! - PCR - Polymerase Chain Reaction Forum

Primers amplify adults but not immatures?! - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-09-2011, 06:02 AM
Pipette Filler
Points: 1,474, Level: 22 Points: 1,474, Level: 22 Points: 1,474, Level: 22
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2009
Posts: 13
Thanks: 1
Thanked 1 Time in 1 Post
Default Primers amplify adults but not immatures?!



Hi I developed species-specific primers for a bark beetle species using the COI gene. The primers amplify the adults but not the larvae. Other primers I developed to amplify other species in the same genues for the COI gene amplify DNA from both larvae and adults.

I am at a complete loss as to the possible cause of this.
Reply With Quote
  #2  
Old 06-09-2011, 07:15 AM
Graduate Student
Points: 3,129, Level: 36 Points: 3,129, Level: 36 Points: 3,129, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2008
Posts: 108
Thanks: 4
Thanked 48 Times in 42 Posts
Default Re: Primers amplify adults but not immatures?!

What is the source of the template you are using? Genomic DNA or cDNA (reverse-transcribed from mRNA)?

If it's mRNA, then you have a potentially nice findings.

If not, well you better post more details on your techniques so that the folks on this board can try to help you out.
Reply With Quote
  #3  
Old 06-09-2011, 05:58 PM
Pipette Filler
Points: 1,474, Level: 22 Points: 1,474, Level: 22 Points: 1,474, Level: 22
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2009
Posts: 13
Thanks: 1
Thanked 1 Time in 1 Post
Default Re: Primers amplify adults but not immatures?!

The source is genomic DNA. I am using DNeasy blood and tissue kits and extracting the DNA according to the manufacturer's protoical for animal tissues using whole adults and larvae. These are further purified using hybrid PVPP and Sepharose 4B columns to remove complex polysaccharides and humic acids.
Reply With Quote
  #4  
Old 06-10-2011, 05:34 AM
Graduate Student
Points: 3,129, Level: 36 Points: 3,129, Level: 36 Points: 3,129, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2008
Posts: 108
Thanks: 4
Thanked 48 Times in 42 Posts
Default Re: Primers amplify adults but not immatures?!

In that case, perhaps there is an error in the extraction part. Perhaps you can try to amplify another on the immature DNA template? The purpose is to ensure that there is genomic DNA there that could be amplified.
Reply With Quote
  #5  
Old 06-13-2011, 04:44 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: Primers amplify adults but not immatures?!

Nebulus, have you check DNA concentration (too low?) and possible PCR inhibitors (such as add both adult and larvae DNA template compare to adult alone is there any decrease in intensity?).
Reply With Quote
  #6  
Old 08-20-2012, 10:43 AM
Pipette Filler
Points: 30, Level: 1 Points: 30, Level: 1 Points: 30, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2012
Posts: 15
Thanks: 0
Thanked 0 Times in 0 Posts
Default Oxygen Plants

Oxyplants India deals mainly with Oxygen plants, Nitrogen plants. We are renowned as the Manufacturer of Oxygen Plants, Nitrogen Plants and Acetylene plants.

Oxygen Plants Manufacturers
Nitrogen Plants Manufacturers
Oxygen Plants
Nitrogen Plants

For more details Visit :- oxyplants.net
Reply With Quote
Reply

Tags
adults , amplify , immatures , primers


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
hygromycin primers Vince Mulholland Protocols and Methods Forum 0 10-09-2007 01:12 PM
hygromycin primers Analia Alet Protocols and Methods Forum 0 10-08-2007 09:24 PM
hygromycin primers SVemuri12@gmail.com Arabidopsis and Plant Biology 0 10-07-2007 10:11 PM
Degenerate Primers c1bl PCR - Polymerase Chain Reaction Forum 0 07-31-2007 02:33 PM
optimizing overlap PCR, ligation-free cloning Maximilian Haeussler Protocols and Methods Forum 0 05-29-2007 01:56 PM


All times are GMT. The time now is 01:45 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14264 seconds with 16 queries